Isolation and purification procedure of vasopeptidase peptide inhibitors

Abstract

The present invention patent refers to the isolation and purification of peptides secreted by serpent venom glands, specifically Bothrops jararaca; to the peptide thus obtained, as well as to the production procedures by genetic engineering techniques in procaryotic and eukaryotic systems; to the engineered peptide thus obtained; to the production of said peptide by chemical synthesis, as well as to the peptide resulting from this chemical processing. It also refers to the utilization of said peptides, obtained by different procedures, in distinct pharmaceutical compositions, and introduced into the organism by a variety of means, in order for them to act as inhibitors of vasopeptidases, and consequently reduce systemic arterial blood pressure, and show local vasodilating action.

1 . A process for the isolation and purification of vasopeptidase peptide inhibitors, showing specificity for the carboxyl site of the angiotensin-converting enzyme, secreted by serpent venom glands (BPPs), particularly  BOTHROPS JARARACA,  or produced endogenously (EVASINS), having vasodilating and anti-hypertensive action, comprising of the following steps: 
 a) providing venom, obtained from a pool of  B. jararaca  venom dissolved in deionized water followed by centrifugation at 1500-2000 rpm for 15 to 20 minutes thereby producing a supernatant; passing the supernatant through a gel-filtration column, equilibrated with 30-50 mM ammonium-acetate buffer, pH 5.0 to pH 6.0, at room temperature;    b) conveying the sample to the top of the column and then eluting the components at a flow rate of 1.0 to 2.0 mL/min;    c) obtaining the absorbance profile for each aliquot at 214 nm in order to define the constitution of the pools    d) partially purifying the components of the pools showing potentiating activity by high performance liquid chromatography, for which 0.1 to 1.0% TFA (trifluoro acid)/H 2 O and acetonitrile/H 2 O (9:1) are used as solvents, and a gradient of 5% to 60% of solvent B, with a flow rate of 0.2 to 0.5 mL/min    e) determining the molecular mass and the primary structure of the bradykinin potentiating peptides by mass spectrometry (ESMS-MS).    
     
     
         2 . The process according to  claim 1 , characterized by the fact that in step (d) an HPLC Merck-Hitachi, model L-6200A is used, with the UV-vis detector set at 214 nm, and the reverse-phase column C-18/Beckman (5μ, 4.6×250 mm).  
     
     
         3 - 22 . (canceled)  
     
     
         23 . A vasopeptidases inhibitor with anti-hipertensive and vasodilating action, characterized by having a formula selected from the group consisting of:  
       
         
           
                 
                 
                 
               
                     
                 
                   I 
                   pp 1 aa 1 aa 2 aa 3 P 4 aa 5 aa 6 P 7 P 8 , 
                     
                 
                     
                 
                   II 
                   pp 1 aa 1 aa 2 aa 3 aa 4 P 5 aa 6 aa 7 P 8 P 9 , 
                 
                     
                 
                   III 
                   pp 1 aa 1 aa 2 aa 3 aa 4 aa 5 P 6 aa 7 aa 8 P 9 P 10 , 
                 
                     
                 
                   IV 
                   pp 1 aa 1 aa 2 aa 3 aa 4 aa 5 aa 6 P 7 aa 8 aa 9 P 10 P 11 , 
                 
                     
                 
                   V 
                   pp 1 aa 1 aa 2 aa 3 aa 4 aa 5 aa 6 aa 7 P 8 aa 9 aa 10 P 11 P 12 , 
                 
                   and 
                 
                     
                 
                   VI 
                   pp 1 aa 1 aa 2 aa 3 aa 4 aa 5 aa 6 aa 7 aa 8 P 9 aa 10 aa 11 P 12 P 13 , 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       where: 
 P is always proline and the remaining amino acids are always L-amino acids and are presented in the following one-letter code;  
 pp 1  is the N-terminus;  
 aa 1  is a non-basic amino acid; aa 2  is a non-acid amino acid;  
 aa 3  is a non-acid amino acid;  
 aa 4  is an amino acid;  
 aa 5  is an amino acid;  
 aa 6  for formula II, aa 7  for formula III, aa 8  for formula IV, and aa 9  for formula V is a non-basic amino acid;  
 aa 6  for formula I, aa 7  for formula II, aa 8  for formula III, aa 9  for formula IV, and aa 10  for formula V are always I or A or T wherein:  
                                             G-glycine   N-asparagine         A-alanine   Q-glutamine         P-proline   D-aspartic acid         V-valine   E-glutamic acid         I-isoleucine   K-lysine         L-leucine   R-arginine         S-serine   F-phenylalanine         T-threonin   H-histidine         W-tryptophane   Y-tyrosine         <E-pyroglutamyl                                                    
 
     
     
         24 . The vasopeptidase inhibitor with vasodilation and anti-hypertensive action, according to  claim 23 , characterized by the fact that D and E are acidic amino acids, K and R are basic amino acids, and F, W, and Y are aromatic amino acids.  
     
     
         25 . The vasopeptidase inhibitor with vasodilation and anti-hypertensive action, according to  claim 23 , characterized by the fact that they are peptides of 8-13 amino acids presenting a general formula, which contains the sequence motif at the carboxyl-terminus of the oligopeptide: 
         P X 1  X 2  P P 
       where X 1  can be any amino acid and X 2  usually is I, and the N-terminal amino acid is blocked, and P is proline.  
     
     
         26 - 33 . (canceled)  
     
     
         34 . The vasopeptidase inhibitors of  claim 23  diluted in a physiogogically acceptable carrier.  
     
     
         35 . (canceled)  
     
     
         36 . A process for using the vasopeptidase inhibitor of  claim 23  in vivo, via trans-mucosa, parenterally, or by injection, and/or intravenously for systemic action or localized action in tissue microcirculation.  
     
     
         37 . A process for using the vasopeptidase inhibitor of  claim 23  in vivo, in dosages varying between approximately 1 g to 10 mg/kg of body weight.  
     
     
         38 . A peptide of Formula I: 
         pp 1 aa 1 aa 2 aa 3 P 4 aa 5 aa 6 P 7 P 8   (I) 
       wherein: 
 (a) pp 1  is the N-terminus of the peptide;  
 (b) aa 1  is a non-basic amino acid;  
 (c) aa 2  is a non-acid amino acid;  
 (d) aa 3  is a non-acid amino acid;  
 (e) P 4  is proline;  
 (f) aa 5  is an amino acid selected from the group consisting of glutamine, asparagine, proline, and glycine;  
 (g) aa 6  is an amino acid selected form the group consisting of isoleucine, alanine, and threonin;  
 (h) P 7  and P 8  are proline.  
 
     
     
         39 . A peptide of Formula II: 
         pp 1 aa 1 aa 2 aa 3 aa 4 P 5 aa 6 aa 7 P 8 P 9   (II) 
       wherein: 
 (a) pp 1  is the N-terminus of the peptide;  
 (b) aa 1  is a non-basic amino acid;  
 (c) aa 2  is a non-acid amino acid;  
 (d) aa 3  is a non-acid amino acid;  
 (e) aa 4  is an amino acid;  
 (f) P 5  is proline;  
 (g) aa 6  is a non-acid amino acid;  
 (h) aa 7  I, is an amino acid selected form the group consisting of isoleucine, alanine, and threonin;  
 (i) P 8  and P 9  are proline.  
 
     
     
         40 . A peptide of Formula III: 
         pp 1 aa 1 aa 2 aa 3 aa 4 aa 5 P 6 aa 7 aa 8 P 9 P 10   (III) 
       wherein: 
 (a) pp 1  is the N-terminus of the peptide;  
 (b) aa 1  is a non-basic amino acid;  
 (c) aa 2  is a non-acid amino acid;  
 (d) aa 3  is a non-acid amino acid;  
 (e) aa 4  is an amino acid;  
 (f) aa 5  is an amino acid selected from the group consisting of glutamine, asparagine, proline, and glycine;  
 (g) P 6  is proline;  
 (h) aa 7  is a non-acid amino acid;  
 (i) aa 8  I, is an amino acid selected form the group consisting of isoleucine, alanine, and threonin;  
 (j) P 9  and P 10  are proline.  
 
     
     
         41 . A peptide of Formula IV: 
         pp 1 aa 1 aa 2 aa 3 aa 4 aa 5 aa 6 P 7 aa 8 aa 9 P 10 P 11   (IV) 
       wherein: 
 (a) pp 1  is the N-terminus of the peptide;  
 (b) aa 1  is a non-basic amino acid;  
 (c) aa 2  is a non-acid amino acid;  
 (d) aa 3  is a non-acid amino acid;  
 (e) aa 4  is an amino acid;  
 (f) aa 5  is an amino acid selected from the group consisting of glutamine, asparagine, proline, and glycine;  
 (g) aa 6  is a non basic amino acid;  
 (h) P 7  is proline;  
 (i) aa 8  is a non-acid amino acid;  
 (j) aa 9  I, is an amino acid selected form the group consisting of isoleucine, alanine, and threonin;  
 (k) P 10  and P 11  are proline.  
 
     
     
         42 . A peptide of Formula V: 
         pp 1 aa 1 aa 2 aa 3 aa 4 aa 5 aa 6 aa 7 P 8 aa 9 aa 10 P 11 P 12   (V) 
       wherein: 
 (a) pp 1  is the N-terminus of the peptide;  
 (b) aa 1  is a non-basic amino acid;  
 (c) aa 2  is a non-acid amino acid;  
 (d) aa 3  is a non-acid amino acid;  
 (e) aa 4  is an amino acid;  
 (f) aa 5  is an amino acid selected from the group consisting of glutamine, asparagine, proline, and glycine;  
 (g) aa 6  is an amino acid;  
 (h) aa 7  is a non basic amino acid;  
 (i) P 8  is proline;  
 (j) aa 9  a non-acid amino acid;  
 (k) aa 10  I, is an amino acid selected form the group consisting of isoleucine, alanine, and threonin;  
 (l) P 11  and P 12  are proline.  
 
     
     
         43 . A peptide of Formula VI: 
         pp 1 aa 1 aa 2 aa 3 aa 4 aa 5 aa 6 aa 7 aa 8 P 9 aa 10 aa 11 P 12 P 13   (VI) 
       wherein: 
 (a) pp 1  is the N-terminus of the peptide;  
 (b) aa 1  is a non-basic amino acid;  
 (c) aa 2  is a non-acid amino acid;  
 (d) aa 3  is a non-acid amino acid;  
 (e) aa 4  is an amino acid;  
 (f) aa 5  is an amino acid selected from the group consisting of glutamine, asparagine, proline, and glycine;  
 (g) aa 6  is an amino acid;  
 (h) aa 7  is an amino acid;  
 (i) aa 8  is a non basic amino acid;  
 (j) P 9  is proline;  
 (k) aa 10  is an amino acid selected from the group consisting of glutamine, asparagine, proline, and glycine;  
 (l) aa 11  I, is an amino acid selected form the group consisting of isoleucine, alanine, and threonin;  
 (m) P 12  and P 13  are proline.  
 
     
     
         44 . The peptide of any one of claims  38  through  43  wherein pp 1  is pyroglutamyl.  
     
     
         45 . The peptide of any one of claims  38  through  43  wherein aa 1  is an amino acid selected from the group consisting of tryptophane, serine, glycine and asparagine.  
     
     
         46 . The peptide of any one of claims  38  through  43  wherein aa 2  is an amino acid selected from the group consisting of proline, glycine, tryptophane, and arginine.  
     
     
         47 . The peptide of any one of claims  38  through  43  wherein aa 3  is an amino acid selected from the group consisting of proline, alanine, arginine, and tryptophane.  
     
     
         48 . The peptide of any one of claims  38  through  43  wherein aa 4  is an amino acid selected from the group consisting of threonin, proline, glycine, histidine, arginine, tryptophane, and glutamic acid.